A- Grain-feeding and E. coli in Cattle

[Kelsey]

Here is a research assignment I did for Extreme Physiology last tri. It is an analysis of an experiment and lab report done in 1998. I think you will find it very interesting - it shows how feeding grain dramatically increases the amount of E. coli in the manure of cattle. I think it mainly applies to confinment, commercial operations, because it is unlikely any of you feed 60-90% grain!
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Grain feeding and the Dissemination of
Acid-Resistant Escherichia coli from Cattle

Francisco Diez-Gonzalez, Todd R Callaway, Menas G Kizoulis, James B Russell: Grain feeding and the Dissemination of Acid-Resistant Escherichia coli from Cattle. Science. Washington: Sep 11, 1998. Vol.281, Iss. 5383; pg. 1666, 3 pgs.


Objective: To define more precisely the role of grain in promoting the growth of acid-resistant E. coli in the rumen and colon of cattle.

(There were two experiments in the study. The first was a more basic trial to find out if there was any substantial change. Since there was, they performed the second, more in-depth and accurate study.)

STUDY 1
Who: 61 mature, non-lactating, 600kg Holstein cows. They were fed on three different diets: 100% grass and hay, 40% hay and 60% grain, and 20% hay and 80% grain.

Methods:
To determine the potential impact of grain feeding on E. coli in cattle, the researchers removed “colonic digesta” from the rectum of cattle that were fed hay, grass, and varying amounts of rolled corn. The pH was then measured, and the number of E. coli was determined. Then, the E. coli samples were exposed to an “acid shock” which mimicked humans’ gastric juice. The acid shock was one hour in a Luria broth of pH 2.0.

Results:


Diet # of animals / Colon pH / Total E. coli count / E. coli count after acid shock
100% grass and hay / 14 / 7.15 / 20,000 / < 1
40% hay and 60% grain / 31 / 6.6 / 6,300,000 / 25,000
20% hay and 80% grain / 16 / 5.4 / 63,000,000 / 250,000
Acid shock – pH mimicking that of gastric juice (pH 2)
E. coli were measured as viable cells per gram.

STUDY 2
Who: Nine mature, non-lactating, 600kg Holstein cows. They were surgically modified so that ruminal contents could be removed directly from the rumen. Three cattle were fed on each diet, so there was a total of nine cattle.

Methods:
The nine cattle were fed every 2 hours with a rotary feeder (10 kg of dry matter per day). The feeds used were medium-quality timothy hay and a grain mixture (89% rolled corn and 11% soybean meal). The diets were 0, 45, and 90% grain with the remainder being hay. The cattle were allowed 14 days to adapt to the diet and then samples were collected for 4 days. Samples of digesta were obtained from both the rumen and the colon. The samples were analyzed for pH, E. coli bacteria count, and volatile fatty acids (acetic, proprionic, and butyric). Then the E. coli in the samples was exposed to an “acid shock” of pH 2.0 for 1 hour.

Results:

RUMEN

Diet / # of Animals / VFA increase / pH / E. coli
100% grass and hay / 3 / not sig. / 6.8 / 2,000
55% hay and 45% grain / 3 / not sig. / 6.5 / 6,300
10% hay and 90% grain / 3 / not sig. / 6.4 / 80,000

COLON

Diet / # of Animals / VFA increase / pH / E. coli survival (AS) / E. coli / % E. coli survival (AS)
100% grass and hay / 3 / 0 / 7.15 / 16,000 / < 2 / 0.01%
55% hay and 45% grain / 3 / 225% / 6.5 / 40,000 / 300 / 1.30%
10% hay and 90% grain / 3 / 400% / 5.0 / 50,000,000 / 5,000,000 / 10%
VFA – Volatile Fatty Acids
E. coli were measured as viable cells per gram.
AS – after acid shock

Limitations:
None.

Applications:
The researchers also found that after cattle had been on a 90% grain diet for some time, when they were switched to an all-hay and grass diet the number of acid-resistant E. coli was reduced to the same as if they had been on an all-hay and grass diet the whole time, after just five days.
The researchers said, “Grain feeding is a practice that promotes the production and efficiency of cattle production, and it is unlikely that American cattle will ever be fed diets consisting only of hay [although currently grassfed beef is becoming widely popular]. However, our studies indicate that cattle could be given hay for a brief period immediately before slaughter to significantly reduce the risk of food-borne E. coli infection.”

Opinion:
This is a very important study, but it seems that the food industry has paid little attention to it. If they were to follow the study’s recommendation of feeding hay to cattle for a week before slaughter, it would definitely help reduce the concentration of disease-causing E. coli bacteria on the beef and subsequently in our food supply.

[Kelsey]

I wrote what I posted the first time; here is the whole study.
By the way, that teacher has climbed Mt Everest twice and drinks Iris milk! Yes, he definitely paid attention!

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Copyright American Association for the Advancement of Science Sep 11, 1998
[Headnote]
Francisco Diez-Gonzalez, Todd R. Callaway, Menas G. Kizoulis, James B. Russell*

[Headnote]
The gastric stomach of humans is a barrier to food-borne pathogens, but Escherichia coli can survive at pH 2.0 if it is grown under mildly acidic conditions. Cattle are a natural reservoir for pathogenic E. coli, and cattle fed mostly grain had lower colonic pH and more acid-resistant E. coli than cattle fed only hay. On the basis of numbers and survival after acid shock, cattle that were fed grain had 106-fold more acid-resistant E. coli than cattle fed hay, but a brief period of hay feeding decreased the acid-resistant count substantially.

Foods can be cooked or irradiated to kill bacteria, but there are ~30 million foodborne illnesses each year in the United States (1). A variety of hypotheses have been formulated to explain the increased incidence of food-borne illness (2, 3). Modern societies tend to "dine out" more often and consume more processed food. Modern detection methods for pathogenic bacteria are more sensitive, and this sensitivity has heightened our awareness of the problem (4). Some experts have suspected a more rapid evolution of bacterial virulence factors, but this evolution is poorly understood (5).

Although Escherichia coli is a normal inhabitant of the gastrointestinal tract, some strains (for example, 0157:H7) produce toxins and are pathogenic (6). Hamburger has frequently been contaminated with pathogenic E. coli, and vegetables and fruit juices have also been sources of infection (4). Cattle, a natural reservoir for pathogenic strains, have often been implicated in E. coli infection (7). It is virtually impossible to prevent all fecal contamination of meat at slaughter, and vegetables are sometimes fertilized with cattle manure. The ability of E. coli to cause foodborne illness is enhanced by its low infective dose, and as few as 10 cells can cause infection (8).

The ability of bacteria to act as food-borne pathogens depends on their capacity to survive the low pH of the gastric stomach and to colonize the intestinal tract of humans (9), but the role of acid resistance in the dissemination of pathogenic bacteria has often been ignored. Pathogenic and nonpathogenic E. coli cultures develop extreme acid resistance only when they are grown at mildly acidic pH. If E. coli is grown at neutral pH, it is acid sensitive and killed by the low pH of gastric juice (10).

Since the Second World War, fattening beef cattle in the United States have been fed large amounts of grain (starch) and very little hay (11), but the impact of grain feeding on acid-resistant E. coli had not been examined. Many forms of starch pass through the pregastric stomach (rumen) to the intestines (II), and cattle are deficient in the starchdegrading enzyme, amylase (12). Starch can be fermented in the colon, and E. coli ferments maltose, an extracellular degradation product of starch (13). Starch fermentation in the colon produces volatile fatty acids (acetate, butyrate, and propionate) that decrease pH (12).

To determine the potential impact of grain feeding on E. coli in cattle, we removed colonic digesta from the rectums of cattle that were fed hay, grass, and varying amounts of rolled com. Digesta were diluted 10-fold with sterile anaerobic water and mixed vigorously with a vortex mixer for 1 min. The pH was measured with a combination electrode. Coliforms were enumerated by visually monitoring turbidity after serial dilution in lauryl sulfate broth (14). Escherichia coli was determined by screening the bacteria on the basis of lactose fermentation, gas production, indole production, the methyl red reaction, Voges-Proskauer test, and citrate fermentation (14). Acid shock was performed by diluting digesta samples 100-fold into Luria broth that had been adjusted to pH 2.0 (14). After 1.0 hour at pH 2.0, viable cell numbers were determined by serially diluting into lauryl sulfate broth.

A survey of 61 cattle indicated that grain supplementation could increase total and acid-resistant E. coli numbers (Table 1). Cattle fed either hay or fresh grass (pasture) had a colonic pH greater than 7.0, the total E. coli count was only 20,000 cells per gram, and virtually all of these bacteria were killed by an acid shock that mimicked the pH of gastric juice. Moderate amounts of grain (60% of dry matter) did not cause a statistically significant decrease in pH (P < 0.05), but the total E. coli population was 6.3 x 10^sup 6^ viable cells per gram of digesta. Some of the E. coli were killed by acid shock, but the acid-resistant count was greater than 25,000 viable cells per gram. When animals were fed more than 80% grain, the pH was significantly lower (P < 0.05), and the acid-resistant E. coli count was 250,000 viable cells per gram.
Table
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Fig 1.
Table 1.

To define more precisely the role of grain in promoting the growth of acid-resistant E. coli, we performed highly controlled experiments. Mature, nonlactating Holstein cows were surgically modified so that ruminal contents could be removed directly from the rumen (IACUC protocol 95-1-97). Cattle of similar size (600 kg) were fed every 2 hours with a rotary feeder (10 kg of dry matter per day). The feeds used were medium-quality timothy hay (14% crude protein, 40% neutral detergent fiber) and a grain mixture [89% rolled (cracked) corn and 11% soybean meal]. The diets were 0, 45, and 90% grain with the remainder being hay.

Samples of digesta were obtained from the rumen as well as the colon. Ruminal contents were squeezed through cheesecloth and purged with oxygen-free carbon dioxide. Colonic samples were processed as described above. Samples were centrifuged at 13,000g for 10 min to remove bacteria and feed particles, and fermentation acids were analyzed by high-pressure liquid chromatography. The total count of anaerobic bacteria was determined by serially diluting the digesta in a nonselective medium designed for strictly anaerobic bacteria (15). Samples of colonic digesta were processed as described above. E. coli strains arising from isolated colonies were obtained from MacConkey's plates supplemented with sorbitol as an energy source. E. coli strains isolated from cattle and E. coli 0157:H7 were given an even longer acid shock (6 hours), and in this case the recovery medium was Luria broth.

The randomized block design was a 3 x 3 Latin square (three animals X three diets) with 14 days of adaptation and 4 days of sample collection (total of 54 days). Because the animals were mature, and the environment of the barn was carefully controlled, the effect of time was judged to be inconsequential. The data were first analyzed by two-way analysis of variance (diet versus animal), and the F values indicated that P < 0.05 in all cases. Student-Newman-Keuls test (16) was used to estimate differences among means, and the variance estimates were pooled (17).

When cattle were fed increasing amounts of grain, the volatile fatty acid (acetic, propionic, and butyric) concentration of the rumen did not increase significantly (P > 0.05), but the concentration in the colon increased -fourfold (P < 0.05) (Fig. lA). Under these conditions, ruminal pH remained essentially constant (P > 0.05), but the pH of the colon decreased (P < 0.05) when the volatile fatty acids accumulated (Fig. lB). Lactic and succinic acids were never detected in rumen samples, but small amounts of both acids were observed in colon samples when 90% grain was fed. Grain supplementation had little effect on the numbers of anaerobic bacteria in the rumen, but the colon count increased 1000-fold (Fig. 2A). Hay-fed cattle had less than 10^sup 5^ colonic coliforms, but those fed 90% grain had ~10^sup 8^ coliforms per gram of digesta (Fig. 2B). Only a small fraction of the ruminal coliforms were E. coli, but virtually all of the colonic coliforms were identified as E. coli (Fig. 2C).

Hay-fed cattle had a low concentration of volatile fatty acids in their colons (Fig. lB), and acid shock killed more than 99.99% of the E. coli (Fig. 3A). When diets were supplemented with either 45 or 90% grain, acids accumulated, colonic pH declined (Fig. 1B), and a much larger percentage of the E. coli survived acid shock (Fig. 3B). The idea that grain, by promoting acid production in the colon, was regulating acid resistance in vivo, was corroborated by in vitro experiments. When E. coli strains isolated from the cattle were grown in the laboratory with a high concentration of glucose, acetic acid accumulated in the medium, pH declined, and the cell survival after acid shock was high (Fig. 3B). If the glucose concentration of the medium was low, little acid was produced, and cell survival was extremely low. Strains isolated from cattle fed forage or grain, and E. coli 0157:H7 (ATCC 43895, CDC EDL 933) behaved similarly, and this result indicated that grain feeding was inducing acid resistance rather than selecting a different population of E. coli.
Graph
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Fig. 2.
Fig. 3.
Fig. 4.

About 5% of our E. coli isolates (n = 155) were sorbitol negative, a diagnostic trait of 0157:H7 (14), but none of these strains tested positive for 0157:H7 antigens (18). The absence of E. coli 0157:H7 in our cattle is not surprising. Previous workers have noted that nonpathogenic E. coli can often outgrow pathogenic strains, and this point is illustrated by at least three observations: (i) The percentage of 0157:H7-positive animals in herds directly linked to outbreaks was less than 2% (19); (ii) even cattle experimentally inoculated with E. coli 0157:H7 did not shed the bacterium for long periods of time (20); and (iii) E. coli 0157:H7 numbers can be reduced by giving animals doses of nonpathogenic E. coli (21).

The finding that grain feeding increased both the number and acid resistance of E. coli in cattle could have significant implications for food safety. Although not all E. coli are pathogenic, there is always the risk that at least some cattle will harbor pathogenic strains. Acid resistance appears to be a factor in the dissemination (transmission) of E. coli from cattle to humans. Therefore, it is reasonable to suggest that the induction of acid resistance could increase the risk of foodborne illness. Our studies indicated that the time needed to decrease E. coli numbers was relatively short (Fig. 4A). Cattle adapted to a 90% grain diet had an acid-resistant E. coli count greater than 106 viable cells per gram. After change to a hay diet, the viable cell number immediately declined, and after 5 days the E. coli population was nearly 106fold lower (Fig. 4B).

Grain feeding is a practice that promotes the production and efficiency of cattle production, and it is unlikely that American cattle will ever be fed diets consisting only of hay. However, our studies indicate that cattle could be given hay for a brief period immediately before slaughter to significantly reduce the risk of food-borne E. coli infection.
[Sidebar]
27 March 1998; accepted 3 August 1998

[Reference]
References

[Reference]
1. Foodborne Pathogens: Risks and Consequences (Center for Agricultural Science and Technology, Task Force Report number 122, CAST, Ames, IA, 1994).
2. R. V. Tauxe, Emerging Infect. Dis. 3, 425 (1997).
3. J. E. Collins, ibid, p. 471.

[Reference]
4. R. L Buchanan and M. P. Doyle, Food Technol. 51, 69 (1997).
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13. E. C. C. Lin, in Escherichia coli and Salmonella Cellular and Molecular Biology, F. C. Neidhardt et al., Eds. (American Society for Microbiology, Washington, DC, ed. 3, 1996), vol. 1, chap. 20.

[Reference]
14. A. D. Hitchins, P. Feng W. D. Watkins, S. R. Rippey, L. A. Chandler, in Food Drug Administration Bacteriological Analytical Manual (Association of Official Analytical Chemists International, Gaithersburg MD, ed. 8, 1995), chap. 4.
15. D. R. Caldwell and M. P. Bryant, Appl. Microbiol. 14,
794 (1966).
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Data Analysis (Wadsworth, Belmont, CA,1993).
18. RIM E. coli 0157:H7 Latex Test (Remel, Lenexa, KS).
19. G. L. Armstrong J. Hollingsworth, J. G. Morris Jr., Epidemiol. Rev. 18, 29 (1996).
20. W. C. Cray and H. W. Moon, Appl. Environ. Microbiol. 61, 1586 (1995).
21. T. Zhao et al., J. Clin. Microbiol. 36, 641 (1998).

[Author Affiliation]
Division of Biological Sciences, Section of Microbiology, Cornell University and Agricultural Research Service, U.S. Department of Agriculture, Ithaca, NY 14853-8101, USA.
*To whom correspondence should be addressed. Email: [e-mail].